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Related Concept Videos

Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
Overview of Electron Microscopy01:25

Overview of Electron Microscopy

The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.
Transmission Electron Microscopy01:15

Transmission Electron Microscopy

In 1931, physicist Ernst Ruska—building on the idea that magnetic fields can direct an electron beam just as lenses can direct a beam of light in an optical microscope—developed the first prototype of the electron microscope. This development led to the development of the field of electron microscopy. In the transmission electron microscope (TEM), electrons are produced by a hot tungsten element and accelerated by a potential difference in an electron gun, which gives them up to 400 keV in...
Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.

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Related Experiment Video

Updated: May 17, 2026

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox
07:12

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

Published on: July 5, 2021

Objective-type total internal reflection microscopy (emission) for single-molecule FRET.

Chirlmin Joo, Taekjip Ha

    Cold Spring Harbor Protocols
    |November 3, 2012
    PubMed
    Summary
    This summary is machine-generated.

    Single-molecule fluorescence resonance energy transfer (smFRET) allows direct observation of biological processes. This protocol details sample preparation and objective-type total internal reflection (TIR) microscopy setup for emission measurements.

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    Last Updated: May 17, 2026

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    Conducting Multiple Imaging Modes with One Fluorescence Microscope
    08:32

    Conducting Multiple Imaging Modes with One Fluorescence Microscope

    Published on: October 28, 2018

    Area of Science:

    • Biophysics
    • Molecular Biology
    • Microscopy

    Background:

    • Single-molecule (sm) detection offers insights into biological events, avoiding population averaging.
    • Förster (fluorescence) resonance energy transfer (FRET) measures distances (30-80 Å) between donor and acceptor molecules.
    • Changes in FRET efficiency signal structural or motion dynamics in biomolecules.

    Purpose of the Study:

    • To describe a protocol for preparing fluorescent bead samples.
    • To detail the setup of objective-type total internal reflection (TIR) microscopy for smFRET emission measurements.

    Main Methods:

    • Preparation of fluorescent bead samples for smFRET analysis.
    • Setting up objective-type total internal reflection (TIR) microscopy for fluorescence detection.
    • Utilizing TIR microscopy for enhanced signal-to-noise ratio in single-molecule studies.

    Main Results:

    • A reproducible method for preparing fluorescent bead samples was established.
    • The protocol provides a clear guide for setting up objective-type TIR microscopy.
    • Successful emission measurements using the described TIR setup are demonstrated.

    Conclusions:

    • Objective-type TIR microscopy is a viable method for single-molecule fluorescence detection.
    • This protocol facilitates advanced studies of molecular dynamics using smFRET.
    • The described setup enables precise distance measurements in biological systems.